Microbial synthesis from aldehyde containing hydrocarbon derived products

ABSTRACT

Proteins, amino acids, gums, and other valuable fermentation products are efficiently biosynthesized from hydrocarbon derived feedstocks containing aldehydes which have been admixed with a nitrogen-containing compound before being passed to a fermentor.

United States Patent [191 Hitzman [111 3,856,774 [451 Dec. 24, 1974 1MICROBIAL SYNTHESIS FROM ALDEHYDE CONTAINING HYDROCARBON DERIVEDPRODUCTS [75] Inventor: Donald 0. IIitzman, Bartlesville,

Okla.

[73] Assignee: Phillips Petroleum Company,

Bartlesville, Okla.

[22] Filed: June 4, 1973 [21] Appl. No.: 366,563

Related U.S. Application Data [60] Continuation-in-part of Ser. No.167,177, July 29, 1971, abandoned, which is a division of'Scr. No.751,926, Aug. 12, 1968, Pat. No. 3,642,578,

[52] U.S. Cl 260/209 R, 195/31 P [51] Int. Cl Cl2d 13/00 [58] Field ofSearch 195/28, 49. 31 P;

Tanaka et a1. 195/28 R Primary ExaminerLione1 M. Shapiro AssistantExaminerR. B. Penland [57] ABSTRACT Proteins, amino acids, gums, andother valuable fermentation products are efficiently biosynthesized fromhydrocarbon derived feedstocks containing aldehydes which have beenadmixed with a nitrogen-containing compound before being passed to afermentor.

Claim 4, 1 Drawing Figure PATENTED [1EE24 I974 FEED PRODUCT NITROGEN JCOMPOUND MICROBIAL SYNTHESIS FROM ALDEHYDE CONTAINING I-IYDROCARBONDERIVED PRODUCTS 1 This is a continuation of application Serial No.167,177, filed July 29, 1971, now abandoned, which latter is adivisional application of application Ser. No. 751,926, filed Aug. 12,1968, now allowed andissued as US. Pat. No. 3,642,578, issued Feb. 15,1972.

This invention relates to a process of microbial con version ofhydrocarbon derived products to proteins, amino acids, gums, and othervaluable products. In another embodiment, this invention relates to amethod of utilizing oxidized derivatives of methane, such asformaldehyde and methanol, as the feedstock for microbial fermentation.This invention further relates to a unique combination of integratedprocedures for microbial synthesis of cellular products.

It is known that microorganisms have the ability to manufacture edibleprotein by the fermentation of hydrocarbons.

There is avid interest in this field for hydrocarbons represent one ofthe greatest sources of raw materials suitable as potential foodstuffsthat can be employed to meet a continuing critical world shortage ofedible protein.

Large-scale protein synthesis has not developed to the high degree ofefficiency wherein high yields of protein are obtained by economicalprocedures.

The conversion of methane and n-paraffins to edible protein isrecognized, but protein so manufactured is known to be frequentlycontaminated by oil and other deleterious hydrocarbons often resultingin decreased cellular yields and high production cost necessitated byextensive separating, centrifuging, and washing procedures in order toachieve an efficacious product free from aromatic and carcinogeniccontamination and hence, suitable as foodstuffs.

Most oxidation products of methane, such as formaldehyde, are consideredbiocidal in nature and deleterious to the microbial fermentationprocess. Commercial methanol, an oxidation product of methane, which canbe used as a hydrocarbon derived feedstock often contains deleteriousquantities of formaldehyde and consequently inhibits or decreasesmicrobial productivity.

It is known that some microorganisms are capable of incorporating andoxidizing formaldehyde but heretofore formaldehyde, even at lowconcentrations, was considered biocidal by those skilled in the art.

In accordance with the instant invention, it has surprisingly beendiscovered that hydrocarbon derived products containing substantialamounts of aldehydes, even formaldehyde, whose toxicity tomicroorganisms is well documented, can be successfully employed as anutritional feedstock for microbial fermentation when they have beenadmixed with a nitrogen-containing compound before being passed to afermentor.

According to one embodiment of this invention, a procedure has beendiscovered for utilizing products formed by the oxidation ofhydrocarbons. Conversion procedures for obtaining the hydrocarbonderived product, such as the Fischer-Tropsch synthesis Topsoe, 1C] orother chemical synthesis processes, can be employed to produce themicrobial feedstock. High quality protein can also be economically andabundantly manufactured by the oxidation and fermentation of widelyavailable hydrocarbon sources such as natural gas, petroleum, naphtha,coal, peat, asphalt and the like.

In another embodiment of the invention, hydrocarbons are oxidized andcontacted with an aldehyde reactive nitrogen-containing compound, andthe watersoluble mixture formed thereby is fed to a fermentor formicrobial fermentation resulting in an uncontam' inated microbialproduction product suitable as a protein food source.

In still another embodiment of this invention, substantial quantities ofprotein are economically synthesized by an integrated process wherein ahydrocarbon is oxidized, the resultant mixture water washed, the aqueoussolution is contacted with ammonia, and the water-soluble mixture isseparated and fed directly into a fermentor.

It is an object of this invention to provide protein, amino acids, andother valuable microbial production products to alleviate the continualworld shortage of foodstuffs. It is an object of this invention toprovide an improved process for the utilization of methane derivativesincluding methanol as the microbial feedstock. It is an object of thisinvention to produce gum fermentation products suitable as adhesives,water viscosifiers, oil recovery adjuncts, etc. It is another object ofthis invention to provide an economical procedure whereby feedstockscontaining aldehydes can be effectively fed directly into a fermentorfor cellular production. Other objects, advantages, embodiments of thisinvention will be evident to those skilled in the art from thedisclosure and the discussion herein set forth.

FIG. 1 is a diagram of a schematicflow sheet demonstratingimplementation of some embodiments of our invention.

Unfortunately, according to the literature, in the direct oxidation ofone of the most abundant hydrocarbon sources available, methane, to theoxidized hydrocarbon derivative methanol, formaldehyde is also produced.The concentration of formaldehyde, furthermore, become biocidal whenconversion rates of methanol that would lend themselves to efficienteconomic production, are employed. utilization of pure methanol is ofteneconomically prohibitive.

This tremendous obstacle to the feasibility of efficiently andeconomically manufacturing synthetic high grades of protein has now beeneliminated. A process has now been discovered whereby nutrienthydrocarbon derived feedstocks containing deleterious quantities ofaldehydes, ketones, carboxylic acids, and the like can effectively beincorporated in the fermentation process by reacting the aldehydecontaining feed with nitrogen containing compounds before the resultantmixture is fed to a fermentor. In accordance with this method, aldehydessuch as formaldehyde or acetaldehyde are not only tendered innocuous butthey can be employed as the sole carbon and energy source for microbialproduction of protein.

A hydrocarbon, such as methane, can now be oxidized to methanol andformaldehyde without using expensive carefully controlled oxidationreaction steps to prevent the formation of deleterious quantities ofaldehydes. Time and expense are consequently, jointly conserved, It isto be understood that when the term by drocarbon derived is used, we arereferring to compounds that can be produced from hydrocarbon such as byoxidation or that can be obtained by other known methods.

Our process has enhanced desirability because higher maximum efficiencyof microbial activity is achieved. the hydrocarbon-derived feedstockthat has previously been partially oxidized and rendered essentiallyfree of oil and other undesirable contaminants permits maximumefficiency and conservation of microbial activity.

Examples of those products which can be employed as microbial feedstocksaccording to this invention include the water-soluble aliphaticalcohols, ketones, al dehydes, carboxylic acids, ethers, and polyols,preferably containing as many as carbon atoms. Some illustrativeexamples include: methanol, ethanol, propanol, butanol, pentanol,hexanol, 1,7-heptanidol, 2-heptanol, 2-methyl-4-pentanol, pentanoicacid, 2- methylbutanoic acid, Z-pentanol, 2-methyl-4-butanol,2-methyl-3-butanol, 2-butanol, 2-methyl-l-propanol, 2-methyl-2-propanol,2-propanol, formic acid, acetic acid, propanoic acid, formaldehyde,acetaldehyde, propanal, butanal, 2-methylpropanal, butanoic acid,2-methylpropanoic acid, pentanoic acid, glutaric acid, hexanoic acid,Z-methylpentanoic acid, heptandioic acid, heptanoic acid, 4-heptanone,Z-heptanone, octanoic acid, 2-ethylhexanoic acid, glycerine, ethyleneglycol, propylene glycol, 2-propanone, 2-butanone, diethyl ether, methylethyl ether, dimethyl ether, di-npropyl ether, n-propyl isopropyl ether,and the like.

Our discovery comprises a process wherein the microbial feed iscontacted with nitrogen-containing compounds that are reactive withaldehydes. The aldehydes modified by or in the presence of thenitrogencontaining compound become effective nutrients and the resultantmixture can be fed directly into a fermentor as a carbon and hydrogennutrient feedstock for cellular production by the microorganisms underconditions suitable for fermentation. It is preferred that only thewater-soluble products of the resultant mixture be fed to the fermentor.

Efficient utilization of almost any hydrocarbon or carbonaceous rawmaterial such as natural gas, petroleum, naphtha, coal, peat, asphaltand the like; and conversion thereof via oxidation and subsequentmicrobial conversionto microbial production products essentially free ofoil and other hydrocarbon contaminants is obtained with an excellentfermentation productivity rate according to this process.

Ethylene can be converted to acetaldehyde and the acetaldehyde and otheroxidized water-soluble products resulting from the oxidation process canbe used as a feedstock after they have been admixed with ammonia ornitrogen-containing compounds such as urea. Since nitrogen is requiredfor cellular growth, both to neutralize the acids produced and toprovide nitrogen for protein synthesis, our method of admixing anitrogen-containing compound such as ammonia or urea prior to thefermentation effectively accomplishes the foregoing in addition torendering the feedstock nontoxic if aldehydes are present. Carbondioxide produced by microbial metabolism in the fermentation process canbe incorporated into the whole process by recycling and converting itfor use in a F ischer-Tropsch synthesis, or other chemical synthesisprocess known to the art, as described in Kirk-Othmer, Encyclopedia ofChemical Technology, 2nd Edition, Vol. 13, pp 382-383, and Vol. 4, pp446480, and consequently promoting increased efficiency.

Other synthetic means of producing the oxygenated hydrocarbon feedsource are likewise familiar to those knowledgeable in the art and canbe produced such as according to the well-known oxo process where asuitable olefin is hydroformylated with carbon monoxide and hydrogen toform aldehydes and alcohols and the Topsoe and ICI synthesis asdescribed in Oil and Gas Journal, Aug. 14, 1967, p 82, and Feb. I2,1968, p 106-109.

FIG. 1 is exemplary of one preferred embodiment of this novel processand represents a schematic flow sheet to aid in the mastery andimplementation of this invention. It is not, however, nor are thematerials used therein, to be construed or interpreted as a limitationon the scope thereof.

In one embodiment of this invention, a crude or impure hydrocarbon orcarbonaceous feed from source 1, can be oxidized in the vessel 2. Theoxidized hydrocarbon derivative can be fed via conduit 3 to the vessel4, wherre it can be admixed with the nitrogen-containing compound fromsource 5. The reaction mixture thereof can be directly fed via conduit 6into the fermentor 7 omitting or by-passing vessel 11, as the microbialfeedstock for cellular production. The discretionary em ployment ofvessel 11 will be discussed hereinafter.

In another variation, an aldehyde-containing feed from said source 1 canbe fed via conduit 9 to said vessel 4 where it can be admixed with saidnitrogencontaining compound from said source 5, and the resultingmixture passed via said conduit 6 directly to said fermentor 7.

Other modifications, such as employing said vessel 2 for theFischer-Tropsch synthesis of oxygenated hydrocarbon derivatives andfeeding the said oxygenated hydrocarbon via said conduit 3 to saidvessel 4 where it is admixed with said nitrogen-containing compound fromsaid source 5, and the resulting mixture passed via said conduit 6directly to said fermentor 7, and the recycling of carbon dioxide fromsaid fermentor 7 via conduit 10 back to said vessel 2 is contemplated.

In still another modification, an oxygenated hydrocarbon from source 1can be fed to said vessel 2 and the nitrogen-containing compound fromsaid source 5 admixed therewith and the resultant mixture fed viaconduit 3 to said vessel 4, water washed therein and the water-solubleproducts thereof fed via said conduit 6 and through the water separationvessel 11 to said fermentor as a water-soluble oxygenated hydrocarbonfeed.

It is believed probable that the conversion of biodeleterious aldehydes,such as formaldehyde, to products such as hexamethylenetetramine and thelike is largely responsible for the microbial suitability of thesematerials as feedstocks.

It is a critical embodiment of our invention that thenitrogen-containing compounds that are reactive with aldehydes beadmixed with the oxygenated hydrocarbon feedstock containing aldehydesbefore introduction of the resultant mixture to the fermentor.

Illustrative examples of suitable nitrogen-containing compounds whichcan be employed include ammonia, ammonium hydroxide, ammonium sulfate,ammonium nitrate, ammonium phosphate, acetonitrile, urea, guanidine,uric acid, and the like. Ammonia or ammonium compounds are presentlypreferred.

Sufficient amounts of the nitrogen-containing compound should be addedto render innocuous a substantial amount of the deleterious material inthe feedstock. Normally, from about 0.01 to 10 mol equivalents of saidnitrogen-containing compound should be provided spheres are used.

One of the most important limitations to increased cell production isthe dissolved oxygen level in the fermentor. The dissolved oxygen can beincreased by running the fermentor under increased pressures. Pressuresof about 0.1 to 50 atmospheres gage are usually employed. Forillustration purposes, the methanol urea solution can be fed to areactor using 8 psig air pressure. The dissolved oxygen level in thefermentor is consequently increased compared to atmospheric pressure andmore cells can be grown in a shorter period of time using identicallysized equipment. In addition, higher temperatures can be maintainedbecause at high pressures the microorganisms can withstand highertemperatures; consequently, cooling expense is reduced. The increasedpressure also aids in the recovery of metabolic products by supplying adriving force for filtration or drum drying. By suddenly releasing thepressure of the fermentor, cells can be ruptured, thus releasing thecellular components and consequently, a product of enhanced purity canbe harvested. The sudden pressure release also volatilizes any volatileimpurities present and enhances the overall efficacy of the process.

Sufficient water is present in the fermentation procedure to provide forthe particular requirements of the microorganisms employed. Generally,any microorganism which is able to utilize oxygenated hydrocarbon feedscan be employed. Suitable hydrocarbon utilizing bacteria can be culturedand developed as follows.

A soil sample is secured from below the ground surface from any desiredplot. Samples of soil taken over a hydrocarbon bearing formation willgenerally contain more hydrocarbon consuming microorganisms than samplesof soil taken over a non-hydrocarbon bearing area. It is preferred thatthe soil sample be taken at a sufficient depth below the surface of theground to avoid surface contamination. Depths ranging from six inches tothree feet are generally preferred, with depths from two to three feetbeing more preferred. When securing the samples, care sould be taken sothat the soil sample be a sample of relatively undisturbed soil at thedesired depth. A convenient method of sampling is to dig a hole with theaid of an ordinary post hole digger to approximately the desired depth;then, by use of a hand auger, take a sample of undisturbed soil from thesite of the hole at the desired depth.

A 200 g sample of soil obtained accordingly is blended for approximately1 minute with 1,000 ml of a sterile medium having the followingcomposition:

The pH of the soil suspension is then adjusted to 7 with anynon-deleterious base while the suspension is agitated. One ml ofthe soilsuspension is then added to ml of the said sterile mineral medium togive a l to 100 dilution soil suspension. One ml of the l to 100dilution is then added to 100 ml of the mineral medium to give a l to10,000 dilution soil suspension. The l to 10,000 soil suspension is then[mixed with sufficient methanol to yield a 5 volume percent mixture. Thecultures are then incubated for 6 days at about 37C, after which streaksare made on Petri dishes containing agar medium prepared using thefollowing recipe:

Sufficient methanol to give 1.5 vol. 71 methanol.

The Petri dishes are incubated for 6 days at about 37C. Viable coloniesare restreaked on other Petri dishes as before to purify the colonies.

Single colonies are then transported to mediums comprised according tothe recipe for Mineral Medium No.. l and containing sufficient methanolto comprise 1.5 volume percent of the total medium.

As will be evident to those skilled in the art various modifications ofthe mineral growth media can be employed thereby resulting in thepropagation of various microorganisms.

The particular microorganism employed in this process is not criticaland we have cultured and usedm any that are suitable for employmentaccording to this invention. Exemplary of said microorganisms arePseudomonas methanica, which has been assigned the numerical designationNRRL 8-3449 by the Northern Utilization Research and DevelopmentDivision, Peoria, 111., Pseudomonasfluorescens, numerical designationNRRL 8-3450, Methanomonas methanica, numerical designation NRRL 8-3450,Methanomonas methanooxidans, numerical designation NRRL 13-3451,Arthobacter parafficum, numerical designation NRRL 8-3453, andCorynebacterium simplex, numerical designation NRRL 8-3454. ThePseudomonas sp. microorganisms were employed through the exemplary runsof our disclosure. Bacillus, Mycobacterium, Actinomyces, and Nocardiagenuses are other illustrate examples of bacteria which have been testedand found to be suitable. Other examples of bacteria include thegenuses: Micrococcus; Rhodobacillus; Chromatium; Nitrosomonas; Serratia;Nitrobacter: Rhizobium; Azotobacter; Aerobacter; Escherichia;Streptococcus; Bactrillum; Clostridium; and Corynebacterium. Othersuitable classes of microorganisms include the yeasts, molds, fungi, andthe like. Combinations of microorganisms can also be employed.

Suitable minerals, growth factors, vitamins, and the like are generallyadded in amounts sufficient to provide for the particular needs of themicroorganisms utilized.

Mineral and growth factors, and. the like, for the microorganisms whichare employed vary according to the particular requirements of themicroorganisms and are generally known to those skilled in the art orare readily determined by those so skilled.

Further addition of nitrogen compounds can be added to the fermentor,such as urea, or ammonia, if desired. The ammonium ions ornitrogen-containing compound charged to the oxidized hydrocarbonfeedstocks of our process are normally a sufficient source of nitrogen,however,

Upon completion of the desired degree of fermentation, the microbialfermentation products can be separated by any means known to the artsuch as centrifugation, filtration, solvent estraction, stripping ofvolatiles, heating, and the like.

We have discovered that the addition of polar organic solvents such asacetone, ethanol, or methanol, after the fermentation has been completedwas surprisingly effectual in precipitating the cells, polymeric gums,and production products from the media. An immediate precipitate wasformed following the addition of excess polar organic solvent and thetightly bound cellular mass could be removed by mechanical means leavinga clear solution from which the solvent could be recovered and recycled.

It is a preferred effect of this invention to produce high qualitynutritionally balanced protein materials suitable as foodstuffs. Inanother embodiment valuable products such as gums, vitamins, aminoacids, growth factors, and the like can be produced.

We have discovered that abnormally high quantities of tryptophane,lysine, leucine, threonine, valine, alanine, and glutamic acid which arenecessary supplements to deficient food can be synthesized according toExample 1 herein after reported by those aforementioned numericallydesignated microorganisms using a methanol-formaldehyde-ammoniumhydroxide feedstock in the mineral salt media. These microorganisms growin a continuous aerobic fermentation process and use this feedstock asboth the carbon and nitrogen source and produce the water-soluble aminoacids tryptophane, lysine, theronine, valine, alanine, and glutamic acidin the media. According to this embodiment, the microbial cells arerecovered and sold as protein and the exhausted culture media isextracted and the amino acids recovered. Identification by paperchromatography establishes that when these aforedesignatedmicroorganisms are grown on a methanol-formaldehyde-ammonium hydroxidefeedstock, essentially these seven said amino acids are produced inabnormally high concentrations and excreted into the media, consequentlyproviding a dual product.

Pseudomonas methanica was particularly high in the production oftryptophane, lysine, and threonine; Pseudomonas fluorescens in theproduction of lysine, threonine, leucine, tryptophane and valine; andCorynebacterium simplex in the production of leucine, lysine, threonine,tryptophane and alanine.

Exemplary of our disclosure and not to be intended as a limitation onthe scope or the materials employed therein, the following examples aregiven.

EXAMPLE I A 14 liter New Brunswick stirred fermentor suitably rigged forcontinuous fermentation and temperature controlled in the range of32-40C, was charged with 7 liters suitable base medium and with 500 ccof the aforesaid inoculum of Pseudomonas sp. (Pseudomonas methanica NRRLB-3449). Materials were charged to the reactor and effluent removeduntil the bacteria had reached an exponential rate of growth and asteady state had been reached. the following data illustratesteady-state fermentor operation (47-70 hours from start-up).

Methanol Consumed Yield of Dried Cells/ lbs ot 72.3 lbs Methane ConsumedPercent Protein of Cells 69.4%

Fermentor Productivity 10.23 g/liter/hr l. EH6 Base Medium has thefollowing amounts of materials per liter of aqueous solution:

KHzPO4 2 K HPO, 2 (NH S0 2 NaCl 0. MgSO,-7H O 3 CaCl 0 Trace MineralsSoln 2. Methanol: Formaldehyde Product is comprised thus:

14 parts Methanol 1 part 37% aqueous HCHO 3. NH,OH was admixed with theMethanolzFormaldehyde Product prior to passing to the fermentor. 4.Trace Mineral solution had the following amounts of the followingcompounds per liter of solution:

5. Assuming 100% of theoretical conversion of CH to CH OH if methane isfirst oxidized to methanol.

Percent protein equals percent N X 6.25.

Fermentor Productivity is in g of dried cells per liter of ferment perhour retention time in fermentor.

Example 1 clearly exemplifies the efficient productivity andhigh-cellular protein content achieved by our process.

EXAMPLE I] A run was effected as in Example 1 except that ammoniumhydroxide was charged to the fermentor separately without prior admixingwith the methanolzformaldehyde feedstock. The culture was eradicated andfermentation ceased.

Example ll demonstrates the criticality of admixing thenitrogen-containing compound of this invention to the oxidizedhydrocarbon containing feedstock prior to passing the feedstock to thefermentor.

Example III A 14 liter New Brunswick fermentor, suitably rigged forcontinuous fermentation and temperature controlled in the range of3240C, was operated at steady-state conditions employing a bacteria asin Example but according to the following conditions:

Base Medium 1.33 liters/hr Trace Minerals 0.005 liter/hr CellConcentration (dry weight) 23 g/litcr (4) See Example I (8) BH-S BaseMedium is comprised as follows:

KH PO, 2.5 g/liter K HPO 2.5 g/Iiter (Ni-[ 1 80 2.0 g/liter NaCl 0.1g/Iiter MgSO -7H O 3.0 g/Iiter CaCl 0.02 g/liter Trace Minerals Solo!3.75 ml/liter The fermentor was monitored by means of: (a) pH of medium;(b) measurement of dissolved O in medium; (c) gas chromatography ofeffluent, e.g., CH OH and HCHO concentration; and (d) measurement ofcell density of medium.

The input of methanol-formaldehyde-ammonium hydroxide and water mixtureand base medium-trace mineral mixture was terminated. Immediately, afeed comprising 14 volume parts CH OH, volume parts HCHO solution (37wt. percent aqueous solution), and 3 volume parts of NH OH solution (25wt. percent aqueous solution) was charged to the fermentor on pH demandso as to maintain the pH at 6.5. A total of 50 cc of this mixture wascharged to the fermentor. Themonitored functions; e.g., pH 6.5, uptakeof O absence of CH OH or HCHO in effluent, and cell density of 23g/liter remained constant. This demonstrated that the ferment wasutilizing the higher HCHO concentration I without difficulty.

When 50 cc of the above feed had been passed to the fermentorthe feedwas terminated. Immediately, a feed comprising 14 volume parts of CH OH,5 volume parts of HCHO solution (37 wt. percent aqueous solution), and 5volume parts of NH OH solution (25 wt. percent A solution) was chargedto the fermentor on pH demand so as to maintain the pH at 6.5. A totalof 160 cc of this mixture was charged to the fermentor: The monitoredfunctions; e.g., pH=6.5, uptake of O absence of CH OH or HCHO ineffluent, and cell density of 23 g/liter remained constant. Thisdemonstrated that the ferment was continuing to utilize the increasedHCHO level of the feed wherein a higher level of NH ,OH was employed.

When 160 cc of the above feed had been passed to the fermentor that feedwas terminated. Immediately, a feed comprising 1 volume part of HCHOsolution (37 wt. percent aqueous solution) and 1 volume part of NH OHsolution (25 wt. percent aqueous solution) was charged to the fermentoron pH demand so as to maintain the pH at 6.5. A total of 85 cc of thismixture was charged to the fermentor. The said monitored functionscontinued to remain constant.

When 85 cc of the above feed had been passed to the fermentor that feedwas terminated. Immediately, a feed essentially comprising 37 wt.percent HCHO aqueous solution was charged to the fermentor on pH demandso as to maintain the pH at 6.5. Almost immediately, the fermentationterminated as was determined by said monitored functions. The culturewas eradicated before 25 cc of HCHO had passed to the fermentor.

Example III demonstrates the utilization of formaldehydes as a carbonenergy source for cellular production by the microorganisms and furtherdemonstrates the criticality of admixing the nitrogen-containingcompound of this invention to the oxidized hydrocarbon containingfeedstock prior to passing feedstock to the fermentor.

As before mentioned, we have discovered that in the fermentation processconducted substantially according to Example I, that considerablequantities of polymeric gums are produced in the product effluent. Thegum can be recovered from the exhausted fermentation media byprecipitation with polar organic solvents. It can then be dried to apowder for convenient storage and use.

Variations in the polymeric gum produced can be achieved by employingvarious of the aforementioned micoorganisms or by variation of themedium composition. Yields of over 50 grams/liter dry weight of gums andcells have been achieved. These polymeric gums can be dissolved in waterto form a solution which has a higher viscosity than pure water.

These gums can be used as water flood additives, as in the recovery ofoil, by forming a solution with a desired viscosity equal that of theoilin-place so that greater efficiency can be achieved in recoveringsaid oil.

The gum can also be used as a drilling mud additive and as a water losscontrol agent. The gum material will increase the viscosity of adrilling mud to which it is added to the desired viscosity and will holdsuspended solids. It has no sensitivity to salts and is compatible withother mud additives.

The polymeric gum can also be used as a selective plugging agent andviscosifier for oil formations, i.e., can be used to increase theviscosity of aqueous compositions used in the production of crude oil.The polymeric gum can be solubilized and injected into the formation ata pH below 10. In this pH range, the polymer penetrates to the desiredzone or acts as a viscosifier. The pH can be adjusted to at least about1 l-l l.5 with a suitable basic material. At this pH, the polymeric gumsare gelled and form a tough, stringy mass. Basic materials can thus beinjected into the formation so that the gum sets up and forms a block.By manipulating the pH, the material can be made very viscous for moreoil recovery or formed into a solid to provide a block. In othersituations, it may be desirable to inject the caustic first and thenfollow it with the soluble gum, using a separate. zone of water ifnecessary so prema ture setting up does not occur except in the zone ofmaximum water penetration. The material is protected from microbialdegradation by the high pH. It is also possible to gel this gum materialby adding to the material agents such as acetone, alcohols, and thelike.

EXAMPLE IV Viscosity of the product effluent containing said polymericgums obtained from a fermentor operated typically as in Example I usingPseudomonasfluorescens NRRL B-3452 was measured in a Brookfield LVTviscosimeter. The viscosities were 14,000 cps, 3800 cps,

11 1,200 cps, 384 cps, and 247 cps at0.3, 1.5, 6.0, 30, and 60 rpm (witha No. 2 spindle), respectively.

The product effluent was diluted with 3 parts of brine for each 1 partof effluent product, and the viscosity of the diluted mixture wasmeasured in the Brookfield viscosimeter employing the No. 2 spindle.Viscosities of 100 cps, 40 cps, 20 cps, and 18 cps were observed at 1.5,6.0, 30.0, and 60.0 rpm, respectively.

The foregoing tests effectively demonstrate that the polymeric gumsproduced according to this invention are useful as viscosifiers forwater and water solutions.

EXAMPLE V The product effluent containing said polymeric gums wasdiluted as above with 3 parts of Burbank brine, brine prepared to havethe salt concentration found in the Burbank Oil Field of Oklahoma, foreach part of product. Sand from formation outcrop known to serve as areservoir for petroleum is packed into a pipe which is 72 inches inlength. The sand is saturated with crude oil from the Burbank Field. Thesand column is then heated to and maintained at the reservoirtemperature of the Burbank Field, while Burbank brine is passed throughthe sand column until no more oil is eluted. Maintaining the temperatureas before, the above I23 mixture of product effluent and Burbank brineis then passed through the sand, until no more oil is eluted. The totalamount of additional oil produced by flooding with the product effluentand brine is equivalent to 7.8 percent of the pore volume of the sandcolumn. Consequently, the foregoing tests demonstrate that flooding withpolymeric gum product effluent and Burbank brine mixture resulted in animproved recovery of petroleum.

The polymeric gum has also been found to possess adhesive qualities andto be suitable as a replacement for casein or soy protein adhesives forcompounding into paper coating agents.

EXAMPLE VI The polymeric gum was employed to bond wood subtrate and wasdiscovered to possess an adhesive lap shear strength of 200 psi basedupon ASTM Test No. D-l002-53T; consequently exemplifying the adhesivecharacteristics of this polymeric gum.

The concepts and products of this invention are applicable for a varietyof useful purposes as indicated throughout our specification. Anothervaluable application of our process is to treat industrial waste andsewage water such as from petroleum and petrochemical operation thatoften contain aldehydes or other toxic contaminants with ammonia orammonium hydroxide and subsequently subject the waste water to microbialaction so as to render the waste material nontoxic. The ammonia treatingstep as taught by our invention permits microbial fermentation of thewaste material by forming an amine compound which is easily degraded bythe microorganism as well as providing favorable pH conditions therefor.

It is within the scope of this invention to vary the organism andfermentation environment to achieve maxium optimum yields of any of themany valuable products such as gums, vitamins, amino acids, growthfactors, and the like that may be desired. Other modifications of thisinvention can be accomplished or followed as will be evident to thoseskilled in the art in light of the foregoing discussion and exampleswithout departing from the spirit and scope thereof.

I claim:

1. A polymeric gum possessing adhesive and water viscosifying qualitiesproduced by a process of microbial synthesis of cellular productionproducts from oxygenated hydrocarbon feedstock containing aldehydes inaddition to other oxygenated hydrocarbons, which process comprises thesteps of:

a. adding to said oxygenated hydrocarbon feedstock containing aldehydesat least one nitrogen.- containing compound reactive with said aldehydeswhereby said aldehydes are rendered substantially innocuous,

b. culturing oxygenated hydrocarbon-utilizing microorganisms on saidnitrogen-containing compound treated feedstock from said step(a),thereby producing said polymeric gum,

wherein said microorganism is selected from the group of genusesPseudomonas, Methanomonas, Arthobacter, Corynebacterium, Bacillus,Mycobacterium, Actinomyoes, Norcardia, Micrococcus, Rhodobacillus,Chromatium, Serratia, Rhizobium, Aerobacter, Escherichia, andStreptococcus. I

2. The polymeric gum of claim 1 wherein said microorganism isPseudomonas methanica NRRL B-3449, Pseudomonas fluorescens NRRL 8-3452.Methanomonas methanica NRRL 8-3450, Metlzunomonus methanoxidans NRRLB-345l. Arrhobacter purafl'icum NRRL 8-3453, or Corynebacterium simplexNRRL B-3454.

3. The polymeric gum of claim 2 wherein said microorganism is saidPseudomonas fluorescens NRRL 8-3452.

4. The polymeric gum of claim 2 wherein said microorganism is saidPseudomonas mezhanicu NRRL 8- 3449.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONPATENT NO. 3,856,774

DATED Decenber 24, 1974 INVENTOR(S) Dcnald O. Hitzman It rs certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Title page, first colum, change "Omtinuatim-in-part" to OcntinuatimSigned and Scaled this fifteenth D y f June 1976 [SEAL] A nest:

C. MARSHALL DANN Commissioner oj'larenrs and Trademarks RUTH C, MASONArresting Officer

1. A POLYMERIC GUM POSSESSING ADHESIVE AND WATER VISCOSIFYING QUALITIESPRODUCED BY A PROCESS OF MICROBIAL SYNTHESIS OF CELLULAR PRODUCTIONPRODUCTS FROM OXYGENATED HYDROCARBON FEEDSTOCK CONTAINING ALDEHYDES INADDITION TO OTHER OXYGENATED HYDROCARBONS, WHICH PROESS COMPRISES THESTEPS OF: A. ADDING TO SAID OXYGENATED HYDROCARBON FEEDSTOCK CONTAININGALDEHYDES AT LEAST ONE NITROGEN:CONTAINING COMPOUND REACTIVE WITH SAIDALDEHYDES WHEREBY SAID ALDEHYDES ARE RENDERED SUBSTANTIALLY INNOCUOUS,B. CULTURING OXYGENATED HYDROCARBON-UTILIZING MICROORGANISMS ON SAIDNITROGEN-CONTAINING COMPOUND TREATED FEEDSTOCK FROM SAID STEP(A),THEREBY PRODUCING SAID POLYMERIC GUM, WHEREIN SAID MICROORGANISM ISSELECTED FROM THE GROUP OF GENUSES PSEUDOMONAS, METHANOMONAS,ARTHOBACTER, CORYNEBACTERIUM, BACILLUS, MYCOBACTERIUM, ACTINOMYOES,NORCARDIA, MICROCOCCUS, RHODOBACILLUS, CHROMATIUM, SERRATIA, RHIZOBIUM,AEROBACTER, ESCHERICHIA, AND STREPTOCOCCUS.